DNA repair by:S.Solali

1. DNA repair by:S.Solali

3. Types of DNA Damage • Deamination: (C  U and A hypoxanthine) • Depurination: purine base (A or G) lost • T-T and T-C dimers: bases become cross- linked, T-T more prominent, caused by UV light (UV-C (<280 nm) and UV-B (280-320 nm) • Alkylation: an alkyl group (e.g., CH3) gets added to bases; chemical induced; some harmless, some cause mutations by mispairing during replication or stop polymerase altogether

5. Types of DNA Damage (cont.) 5. Oxidative damage: guanine oxidizes to 8-oxo-guanine, also cause SS and DS breaks, very important for organelles 6. Replication errors: wrong nucleotide (or modified nt) inserted 7. Double-strand breaks (DSB): induced by ionizing radiation, transposons, topoisomerases, homing endonucleases, mechanical stress on chromosomes, or a single-strand nick in a single-stranded region (e.g., during replication and transcription)

6. IMPORTANCE OF DNA REPAIR Hoeijmakers , 2001

7. DNA Repair • DNA damage may arise: (i) spontaneously, (ii) environmental exposure to mutagens, or (iii) cellular metabolism. • DNA damage may be classified as: (I) strand breaks, (ii) base loss (AP site), (iii) base damages, (iv) adducts, (v) cross-links, (vi) sugar damages, (vii) DNA-protein cross links. • DNA damage, if not repaired, may affect replication and transcription, leading to mutation or cell death.

10. Methyl-directed mismatch repair • If any mismatch escapes the proof reading mechanisms it will cause distortion of the helix. • This can be detected and repaired but it is important that the repair enzyme can distinguish the new strand from the old. • This is possible in E. coli because there is an enzyme which methylates the A in a sequence GATC. This methylation does not occur immediately after synthesis and until it does the two strands are distinguishable.

11. Mismatch repair • MMR system is an excision/resynthesis system that can be divided into 4 phases: • (i) recognition of a mismatch by MutS proteins, • (ii) recruitment of repair enzymes • (iii) excision of the incorrect sequence, • (iv) resynthesis by DNA polymerase using the parental strand as a template.

12. Mismatch Repair in E.coli • MutS is responsible for initiation of E. coli mismatch repair. • 95 kDa polypeptide, which exists as an equilibrium mixture of dimers and tetramers • recognizes mismatched base pairs. • MutL, a 68 kDa polypeptide that is dimeric in solution, is recruited to the heteroduplex in a MutS- and ATP-dependent fashion. • The MutL‚ MutS‚heteroduplex complex is believed to be a key intermediate in the initiation of mismatch repair

13. Methyl Directed MisMatch repair in E. coli

14. Methylataion and Mismatch Repair

15. Model for Mismatch Repair

17. Excision Repair • Conserved throughout evolution, found in all prokaryotic and eukaryotic organisms • Three step process: – 1. Error is recognized and enzymatically clipped out by a nuclease that cleaves the phosphodiester bonds (uvr gene products operate at this step) – 2. DNA Polymerase I fills in the gap by inserting the appropriate nucleotides – 3. DNA Ligase seals the gap

18. Excision Repair • Two know types of excision repair – Base excision repair (BER) • corrects damage to nitrogenous bases created by the spontaneous hydrolysis of DNA bases as well as the hydrolysis of DNA bases caused by agents that chemically alter them – Nucleotide excision repair (NER) • Repairs “bulky” lesions in DNA that alter or distort the regular DNA double helix • Group of genes (uvr) involved in recognizing and clipping out the lesions in the DNA • Repair is completed by DNA pol I and DNA ligase

19. Base excision Repair • For correction of specific Chemical Damage in DNA • Uracil • Hypoxanthine • 3-m Adenine • Urea • Formamidopyrimidine • 5,6 Hydrated Thymine

20. Base excision repair. • Consist of DNA glycosylases and AP endonuclease • The DNA glycosylases are specific • Uracil glycosylase • Hypoxanthine DNA glycosylase • Etc…

21. Mechanism 1.DNA glycosylase recognizes Specific Damaged base 2. Cleaves glycosl bond to remove Base 3. AP endonuclease cleaves Backbone 4. DNA Pol removes abasic site 5. Replacement of Base

22. Deaminated C Base Excision Repair (BER) Variety of DNA glycosylases, for different types of damaged bases. AP endonuclease recognizes sites with a missing base; cleaves sugar-phosphate backbone. Deoxyribose phosphodiesterase removes the sugar-phosphate lacking the base. Fig. 6.15

23. Nucleotide Excision Repair • Used by the cell for bulky DNA damage • Non specific DNA damage • Chemical adducts … • UV photoproducts First identified in 1964 in E.coli. Ludovic C. J. Gillet and Orlando D. Scharer Molecular Mechanisms of Mammalian Global Genome Nucleotide Excision Repair Chem. Rev. 2006, 106, 253-276

24. Excision repair In this form of repair the gene products of the E. coli uvrA, uvrB and uvrC genes form an enzyme complex that physically cuts out (excises the damged strand containing the pyrimidine dimers. An incision is made 8 nucleotides (nt) away for the pyrimidine dimer on the 5’ side and 4 or 5 nt on the 3’ side.. The damaged strand is removed by uvrD, a helicase and then repaired by DNA pol I and DNA ligase. Is error-free.

25. T T Excision Repair in E.coli Damage recognised by UvrABC, nicks made on both sides of dimer 5’ 3’ 3’ 5’ T T 5’ 3’ 3’ 5’ T T Dimer removed by UvrD, a helicase 3’ 5’ 5’ 3’ Gap filled by DNA pol I and the nick sealed by DNA ligase 3’ 5’ 5’ 3’

26. Nucleotide-Excision Repair in E. coli and Humans

27. Excision repair The UvrABC complex is referred to as an exinuclease. UvrAB proteins identify the bulky dimer lesion, UvrA protein then leaves, and UvrC protein then binds to UvrB protein and introduces the nicks on either side of the dimer. In man there is a similar process carried out by 2 related enzyme complexes: global excision repair and transcription coupled repair. Several human syndromes deficient in excision repair, Xeroderma pigmentosum, Cockayne Syndrome, and are characterised by extreme sensitivity to UV light (& skin cancers)

28. Nucleotide Excision Repair • Defects cause • Xeroderma Pigmentosum • 1874, when Moriz Kaposi used this term for the first time to describe the symptoms observed in a patient.13 XP patients exhibit an extreme sensitivity to sunlight and have more than 1000-fold increased risk to develop skin cancer, especiallyin regions exposed to sunlight such as hands, face, neck • Cockayne Syndrome • Trichothiodystrophy

29. Nucleotide Excision Repair • Defects cause • Cockayne Syndrome • A second disorder with UV sensitivity was reported by Edward Alfred Cockayne in 1936. Cockayne syndrome CS) is characterized by additional symptoms such as short stature, severe neurological abnormalities caused by dysmyelination, bird-like faces, tooth decay, and cataracts. CS patients have a mean life expectancy of 12.5 years but in contrast to XP do not show a clear predisposition to skin cancer. CS cells are deficient in transcription-coupled NER but are proficient in global genome NER. • Trichothiodystrophy

30. Nucleotide Excision Repair • Defects cause • Trichothiodystrophy • A third genetic disease characterized by UV sensitivity, trichothiodystrophy (TTD, literally: “sulfur-deficient brittle hair”), was reported by Price in 1980. In addition to symptoms shared with CS patients, TTD patients show characteristic sulfur-deficient, brittle hair and scaling of skin. This genetic disorder is now known to correlate with mutations in genes involved in NER (XPB, XPD, and TTDA genes). All of these genes are part of the 10-subunit transcription/repair factor TFIIH, and TTD is likely to reflect an impairment of transcriptional transactions rather than regular defect in DNA repair. This disorder is therefore sometimes referred as a “transcriptional syndrome”.

31. Photoactivation Repair in E. coli • Exposing UV treated cells to blue light results in a reversal of the thymine dimer formation • Enzyme, photoactivation repair enzyme (PRE) absorbs a photon of light (from blue light) and is able to cleave the bond forming the thymine dimer. • Once bond is cleaved, DNA is back to normal

32. Direct Repair: Photoreactivation by photolyase

33. Alkylation of DNA by alkylating agents

34. O6-methyl G, if not repaired, may produce a mutation

35. Direct Repair:Reversal of O6 methyl G to G by methyltransferase

36. Direct repair of alkylated bases by AlkB. Direct re

38. Pairing of homologous chromosomes and crossing-over in meiosis.

39. Helicase and nuclease activities of the RecBCD

40. Helicase and nuclease activities of the RecBCD

41. The RecBCD pathway of recombination

42. Recombination during meiosisis initiated by double-strand breaks.

43. RuvA and RuvB • DNA helicase that catalyzes branch migration • RuvA tetramer binds to HJ (each DNA helix between subunits) • RuvB is a hexamer ring, has helicase & ATPase activity • 2 copies of ruvB bind at the HJ (to ruvA and 2 of the DNA helices) • Branch migration is in the direction of recA mediated strand-exchange

45. RuvC bound to Holliday junction Fig. 22.31a

46. Models for recombinational DNA repair

47. Models for recombinational DNA repair of stalled replication fork

48. DNA non-homologous end-joining (NHEJ) • Predominant mechanism for DSB repair in mammals. • Also exists in single-celled eukaryotes, e.g. Saccharomyces cerevisiae • Particularly important in G0/G1

49. DNA-PKcs Homologous recombination Non-homologous end-joining DSB DSB Rad50, Mre11, Xrs2 complex Resection Ku70, Ku80 Rad52 Rad50, Mre11, Xrs2 complex Strand invasion “Cleaning up” of ends Rad51; BRCA2 DNA synthesis     XRCC4/Ligase IV Ligation Ligation, branch migration, Holliday junction resolution

50. DNA-PK DNA-PK DNA-dependent protein kinase (DNA-PK) DNA ACTIVE KINASE INACTIVE